Yeast strain for production of four carbon alcohols

ABSTRACT

Yeast cells with a reduced general control response to amino acid starvation were found to have increased tolerance to butanol in the growth medium. The reduced response was engineered by genetic modification of a gene involved in the response, a GCN gene, to eliminate activity of the encoded protein. Yeast strains with an engineered butanol biosynthetic pathway and a genetic modification in a gene involved in the general control response to amino acid starvation, which have increased butanol tolerance, are useful for production of butanol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims the benefit of U.S.application Ser. No. 12/435,530, filed May 5, 2009, which claims thebenefit of U.S. Provisional Applications 61/052,286 and 61/052,289, bothfiled May 12, 2008. Each of the referenced applications is hereinincorporated by reference.

FIELD OF INVENTION

The invention relates to the field of microbiology and geneticengineering. More specifically, yeast genes involved in response tobutanol were identified. Yeast strains with reduced expression of theidentified genes were found to have improved growth yield in thepresence of butanol.

BACKGROUND OF INVENTION

Butanol is an important industrial chemical, useful as a fuel additive,as a feedstock chemical in the plastics industry, and as a foodgradeextractant in the food and flavor industry. Each year 10 to 12 billionpounds of butanol are produced by petrochemical means and the need forthis commodity chemical will likely increase.

Methods for the chemical synthesis of butanols are known, however theseprocesses use starting materials derived from petrochemicals, aregenerally expensive, and are not environmentally friendly. Methods ofproducing butanol by fermentation are also known, where the most popularprocess produces a mixture of acetone, 1-butanol and ethanol and isreferred to as the ABE processes (Blaschek et al., U.S. Pat. No.6,358,717). Acetone-butanol-ethanol (ABE) fermentation by Clostridiumacetobutylicum is one of the oldest known industrial fermentations, andthe pathways and genes responsible for the production of these solventshave been reported (Girbal et al., Trends in Biotechnology 16:11-16(1998)). Isobutanol is produced biologically as a by-product of yeastfermentation. It is a component of “fusel oil” that forms as a result ofincomplete metabolism of amino acids by this group of fungi. Isobutanolis specifically produced from catabolism of L-valine. After the aminegroup of L-valine is harvested as a nitrogen source, the resultingα-keto acid is decarboxylated and reduced to isobutanol by enzymes ofthe so-called Ehrlich pathway (Dickinson et al., J. Biol. Chem.273(40):25752-25756 (1998)). Yields of fusel oil and/or its componentsachieved during beverage fermentation are typically low.

Additionally, recombinant microbial production hosts, expressing a1-butanol biosynthetic pathway (Donaldson et al., copending and commonlyowned U.S. Patent Application Publication No. 20080182308), a 2-butanolbiosynthetic pathway (Donaldson et al., copending and commonly ownedU.S. Patent Application Publication Nos. US 20070259410A1 and US2007-0292927), and an isobutanol biosynthetic pathway (Maggio-Hall etal., copending and commonly owned U.S. Patent Application PublicationNo. US 20070092957) have been described.

Biological production of butanols is believed to be limited by butanoltoxicity to the host microorganism used in fermentation for butanolproduction. Strains of Clostridium that are tolerant to 1-butanol havebeen isolated by chemical mutagenesis (Jain et al. U.S. Pat. No.5,192,673; and Blaschek et al. U.S. Pat. No. 6,358,717), overexpressionof certain classes of genes such as those that express stress responseproteins (Papoutsakis et al. U.S. Pat. No. 6,960,465; and Tomas et al.,Appl. Environ. Microbiol. 69(8):4951-4965 (2003)), and by serialenrichment (Quratulain et al., Folia Microbiologica (Prague)40(5):467-471 (1995); and Soucaille et al., Current Microbiology14(5):295-299 (1987)). Desmond et al. (Appl. Environ. Microbiol.70(10):5929-5936 (2004)) report that overexpression of GroESL, twostress responsive proteins, in Lactococcus lactis and Lactobacillusparacasei produced strains that were able to grow in the presence of0.5% volume/volume (v/v) [0.4% weight/volume (w/v)] 1-butanol.Additionally, the isolation of 1-butanol tolerant strains from estuarysediment (Sardessai et al., Current Science 82(6):622-623 (2002)) andfrom activated sludge (Bieszkiewicz et al., Acta Microbiologica Polonica36(3):259-265 (1987)) has been described. Butanol tolerant bacterialstrains have been isolated from microbial consortia (copending andcommonly owned U.S. Patent Publication Nos. 20070259411, 20080124774 and20080138870) or by mutant screening (copending and commonly owned U.S.patent application Ser. Nos. 12/330,530, 12/330,531, and 12/330,534).

There remains a need for butanol producing yeast strains that are moretolerant to butanols, as well as methods of producing butanols usingyeast host strains that are more tolerant to these chemicals andengineered for butanol production.

SUMMARY OF THE INVENTION

The invention provides a recombinant yeast host which produces butanoland comprises a genetic modification that results in reduced response inthe general control response to amino acid starvation. Such cells havean increased tolerance to butanol as compared with cells that lack thegenetic modification. Reduction in response in the general controlresponse to amino acid starvation may be accomplished via mutation ofendogenous genes that impact the response. Host cells of the inventionmay produce butanol naturally or may be engineered to do so via anengineered pathway.

Accordingly, the invention provides a recombinant yeast host cellproducing butanol where the yeast cell comprises at least one geneticmodification which reduces the response in the general control responseto amino acid starvation.

In one embodiment the yeast cell of the invention comprises a geneticmodification in a gene encoding a protein selected from Gcn1p, Gcn2p,Gcn3p, Gcn4p, Gcn5p, and Gcn20p.

In another embodiment the yeast cell comprises a recombinantbiosynthetic pathway selected from the group consisting of:

a) a 1-butanol biosynthetic pathway;

b) a 2-butanol biosynthetic pathway; and

c) an isobutanol biosynthetic pathway.

In another embodiment the invention provides a method for the productionof butanol comprising the steps of:

-   -   (a) providing a recombinant yeast host cell which        -   1) produces butanol and        -   2) comprises at least one genetic modification which reduces            the response in the general control response to amino acid            starvation; and    -   (b) culturing the strain of (a) under conditions wherein butanol        is produced.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS

The various embodiments of the invention can be more fully understoodfrom the following detailed description, the figures, and theaccompanying sequence descriptions, which form a part of thisapplication.

FIG. 1 shows fractional growth yields of wild type, mutant GCN2 andmutant GCN4 strains at 8 hr (A) and 24 hr (B) time points for growth inYVCM containing different concentrations of isobutanol.

FIG. 2 shows fractional growth yields of wild type, mutant GCN2 andmutant GCN4 strains at 7 hr (A) and 23 hr (B) time points for growth inYPD containing different concentrations of isobutanol.

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions which form a partof this application.

The following sequences conform with 37 C.F.R. 1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

TABLE 1 Summary of Gene and Protein SEQ ID Numbers for 1-ButanolBiosynthetic Pathway SEQ ID NO: SEQ ID NO: Description Nucleic acidPeptide Acetyl-CoA acetyltransferase thlA from 1 2 Clostridiumacetobutylicum ATCC 824 Acetyl-CoA acetyltransferase thlB from 3 4Clostridium acetobutylicum ATCC 824 Acetyl-CoA acetyltransferase from 3940 Saccharomyces cerevisiae 3-Hydroxybutyryl-CoA dehydrogenase 5 6 fromClostridium acetobutylicum ATCC 824 Crotonase from Clostridiumacetobutylicum 7 8 ATCC 824 Putative trans-enoyl CoA reductase from 9 10Clostridium acetobutylicum ATCC 824 Butyraldehyde dehydrogenase from 1112 Clostridium beijerinckii NRRL B594 1-Butanol dehydrogenase bdhB from13 14 Clostridium acetobutylicum ATCC 824 1-Butanol dehydrogenase 15 16bdhA from Clostridium acetobutylicum ATCC 824

TABLE 2 Summary of Gene and Protein SEQ ID Numbers for 2-ButanolBiosynthetic Pathway SEQ ID NO: SEQ ID NO: Description Nucleic acidPeptide budA, acetolactate decarboxylase from 17 18 Klebsiellapneumoniae ATCC 25955 budB, acetolactate synthase from Klebsiella 19 20pneumoniae ATCC 25955 budC, butanediol dehydrogenase from 21 22Klebsiella pneumoniae IAM1063 pddA, butanediol dehydratase alpha subunit23 24 from Klebsiella oxytoca ATCC 8724 pddB, butanediol dehydratasebeta subunit 25 26 from Klebsiella oxytoca ATCC 8724 pddC, butanedioldehydratase gamma 27 28 subunit from Klebsiella oxytoca ATCC 872 sadH,2-butanol dehydrogenase from 29 30 Rhodococcus ruber 219

TABLE 3 Summary of Gene and Protein SEQ ID Numbers for IsobutanolBiosynthetic Pathway SEQ ID NO: SEQ ID NO: Description Nucleic acidPeptide Klebsiella pneumoniae budB (acetolactate 19 20 synthase)Bacillus subtilis alsS 41 42 (acetolactate synthase) E. coli ilvC(acetohydroxy acid 31 32 reductoisomerase) S. cerevisiae ILV5 43 44(acetohydroxy acid reductoisomerase) B. subtilis ilvC (acetohydroxy acid45 46 reductoisomerase) E. coli ilvD (acetohydroxy acid 33 34dehydratase S. cerevisiae ILV3 47 48 (Dihydroxyacid dehydratase)Lactococcus lactis kivD (branched-chain α- 35 36 keto aciddecarboxylase), codon optimized E. coli yqhD (branched-chain alcohol 3738 dehydrogenase)

TABLE 4 Summary of Gene and Protein SEQ ID Numbers for members ofgeneral control system for amino acid biosynthesis SEQ ID NO: SEQ ID NO:Description Nucleic acid Peptide GCN1 from Saccharomyces cerevisiae 4950 GCN2 from Saccharomyces cerevisiae 51 52 GCN3 from Saccharomycescerevisiae 53 54 GCN4 from Saccharomyces cerevisiae 55 56 GCN5 fromSaccharomyces cerevisiae 57 58 GCN20 from Saccharomyces cerevisiae 59 60GCN1 from Yarrowia lipolytica 61 62 GCN2 from Yarrowia lipolytica 63 64GCN3 from Yarrowia lipolytica 65 66 GCN5 from Yarrowia lipolytica 67 68GCN2 from Candida albicans 69 70 GCN3 from Candida albicans 71 72 GCN5from Candida albicans-1 73 74 GCN5 from Candida albicans-2 75  74* *thesame amino acid sequence is encoded by both SEQ ID NO: 73 and 75

SEQ ID NO:76 is the nucleotide sequence of the GPD promoter described inExample 2.

SEQ ID NO:77 is the nucleotide sequence of the CYC1 terminator describedin Example 2.

SEQ ID NO:78 is the nucleotide sequence of the FBA promoter described inExample 2.

SEQ ID NO:79 is the nucleotide sequence of ADH1 promoter described inExample 2.

SEQ ID NO:80 is the nucleotide sequence of ADH1 terminator described inExample 2.

SEQ ID NO:81 is the nucleotide sequence of GPM promoter described inExample 2.

SEQ ID NOs:82-137 are the nucleotide sequences of oligonucleotidecloning, screening or sequencing primers used in the Examples describedherein.

SEQ ID NO:138 is the nucleotide sequence of the “URA3 repeats” fragment.

SEQ ID NOs:139 and 140 are the nucleotide sequences of PCR primers usedto amplify a DNA fragment for gcn2 deletion.

SEQ ID NOs:141 and 142 are the nucleotide sequences of PCR primers usedto amplify a DNA fragment for gcn4 deletion.

SEQ ID NOs:143 and 144 are primer binding sequences that bound directrepeats flanking URA3⁺: in the “URA3 repeats” fragment. SEQ ID NOs:145and 146 are direct repeat sequences that flank the promoter and codingsequence in the “URA3 repeats” fragment.

SEQ ID NO:147 is the promoter sequence in the “URA3 repeats” fragment.

SEQ ID NO:148 is the URA3 coding sequence in the “URA3 repeats”fragment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a recombinant yeast host which producesbutanol and comprises a genetic modification that results in a reducedresponse in the general control response to amino acid starvation. Suchcells have an increased tolerance to butanol as compared with cells thatlack the genetic modification. A tolerant yeast strain of the inventionhas at least one genetic modification that causes the reduced generalcontrol response to amino acid starvation. This reduced response may beaccomplished via mutation of endogenous genes that impact the response.Host cells of the invention may produce butanol naturally or may beengineered to do so via an engineered pathway.

Butanol produced using the present strains may be used as an alternativeenergy source to fossil fuels. Fermentative production of butanolresults in less pollutants than typical petrochemical synthesis.

The following abbreviations and definitions will be used for theinterpretation of the specification and the claims.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, are intended to cover a non-exclusive inclusion. Forexample, a composition, a mixture, process, method, article, orapparatus that comprises a list of elements is not necessarily limitedto only those elements but may include other elements not expresslylisted or inherent to such composition, mixture, process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive or and not to an exclusive or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the indefinite articles “a” and “an” preceding an element orcomponent of the invention are intended to be nonrestrictive regardingthe number of instances (i.e. occurrences) of the element or component.Therefore “a” or “an” should be read to include one or at least one, andthe singular word form of the element or component also includes theplural unless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is anon-limiting term and is not intended to refer to any single embodimentof the particular invention but encompasses all possible embodiments asdescribed in the specification and the claims.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates oruse solutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about”, the claims include equivalents to the quantities. Inone embodiment, the term “about” means within 10% of the reportednumerical value, preferably within 5% of the reported numerical value.

The term “butanol” as used herein, refers to 1-butanol, 2-butanol,isobutanol, or mixtures thereof.

The terms “butanol tolerant yeast strain” and “tolerant” when used todescribe a modified yeast strain of the invention, refers to a modifiedyeast that shows better growth in the presence of butanol than theparent strain from which it is derived.

The term “butanol biosynthetic pathway” refers to an enzyme pathway toproduce 1-butanol, 2-butanol, or isobutanol.

The term “1-butanol biosynthetic pathway” refers to an enzyme pathway toproduce 1-butanol from acetyl-coenzyme A (acetyl-CoA).

The term “2-butanol biosynthetic pathway” refers to an enzyme pathway toproduce 2-butanol from pyruvate.

The term “isobutanol biosynthetic pathway” refers to an enzyme pathwayto produce isobutanol from pyruvate.

The term “acetyl-CoA acetyltransferase” refers to an enzyme thatcatalyzes the conversion of two molecules of acetyl-CoA toacetoacetyl-CoA and coenzyme A (CoA). Preferred acetyl-CoAacetyltransferases are acetyl-CoA acetyltransferases with substratepreferences (reaction in the forward direction) for a short chainacyl-CoA and acetyl-CoA and are classified as E.C. 2.3.1.9 [EnzymeNomenclature 1992, Academic Press, San Diego]; although, enzymes with abroader substrate range (E.C. 2.3.1.16) will be functional as well.Acetyl-CoA acetyltransferases are available from a number of sources,for example, Escherichia coli (GenBank Nos: NP_(—)416728, NC_(—)000913;NCBI (National Center for Biotechnology Information) amino acidsequence, NCBI nucleotide sequence), Clostridium acetobutylicum (GenBankNos: NP_(—)349476.1 (SEQ ID NO:2), NC_(—)003030; NP_(—)149242 (SEQ IDNO:4), NC_(—)001988), Bacillus subtilis (GenBank Nos: NP_(—)390297,NC_(—)000964), and Saccharomyces cerevisiae (GenBank Nos: NP_(—)015297,NC_(—)001148 (SEQ ID NO:39)).

The term “3-hydroxybutyryl-CoA dehydrogenase” refers to an enzyme thatcatalyzes the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA.3-Hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamide adeninedinucleotide (NADH)-dependent, with a substrate preference for(S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and are classifiedas E.C. 1.1.1.35 and E.C. 1.1.1.30, respectively. Additionally,3-hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamide adeninedinucleotide phosphate (NADPH)-dependent, with a substrate preferencefor (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and areclassified as E.C. 1.1.1.157 and E.C. 1.1.1.36, respectively.3-Hydroxybutyryl-CoA dehydrogenases are available from a number ofsources, for example, C. acetobutylicum (GenBank NOs: NP_(—)349314 (SEQID NO:6), NC_(—)003030), B. subtilis (GenBank NOs: AAB09614, U29084),Ralstonia eutropha (GenBank NOs: ZP_(—)0017144, NZAADY01000001,Alcaligenes eutrophus (GenBank NOs: YP_(—)294481, NC_(—)007347), and A.eutrophus (Gen Bank NOs: P14697, J04987).

The term “crotonase” refers to an enzyme that catalyzes the conversionof 3-hydroxybutyryl-CoA to crotonyl-CoA and H₂O. Crotonases may have asubstrate preference for (S)-3-hydroxybutyryl-CoA or(R)-3-hydroxybutyryl-CoA and are classified as E.C. 4.2.1.17 and E.C.4.2.1.55, respectively. Crotonases are available from a number ofsources, for example, E. coli (GenBank NOs: NP_(—)415911 (SEQ ID NO:8),NC_(—)000913), C. acetobutylicum (GenBank NOs: NP_(—)349318,NC_(—)003030), B. subtilis (GenBank NOs: CAB13705, Z99113), andAeromonas caviae (GenBank NOs: BAA21816, D88825).

The term “butyryl-CoA dehydrogenase”, also called trans-enoyl CoAreductase, refers to an enzyme that catalyzes the conversion ofcrotonyl-CoA to butyryl-CoA. Butyryl-CoA dehydrogenases may beNADH-dependent or NADPH-dependent and are classified as E.C. 1.3.1.44and E.C. 1.3.1.38, respectively. Butyryl-CoA dehydrogenases areavailable from a number of sources, for example, C. acetobutylicum(GenBank NOs: NP_(—)347102 (SEQ ID NO:10), NC_(—)003030), Euglenagracilis (GenBank NOs: Q5EU90, AY741582), Streptomyces collinus (GenBankNOs: AAA92890, U37135), and Streptomyces coelicolor (GenBank NOs:CAA22721, AL939127).

The term “butyraldehyde dehydrogenase” refers to an enzyme thatcatalyzes the conversion of butyryl-CoA to butyraldehyde, using NADH orNADPH as cofactor. Butyraldehyde dehydrogenases with a preference forNADH are known as E.C. 1.2.1.57 and are available from, for example,Clostridium beijerinckii (GenBank NOs: AAD31841 (SEQ ID NO:12),AF157306) and C. acetobutylicum (GenBank NOs: NP_(—)149325,NC_(—)001988).

The term “1-butanol dehydrogenase” refers to an enzyme that catalyzesthe conversion of butyraldehyde to 1-butanol. 1-butanol dehydrogenasesare a subset of the broad family of alcohol dehydrogenases. 1-butanoldehydrogenase may be NADH- or NADPH-dependent. 1-butanol dehydrogenasesare available from, for example, C. acetobutylicum (GenBank NOs:NP_(—)149325, NC_(—)001988; NP_(—)349891 (SEQ ID NO:14), NC_(—)003030;and NP_(—)349892 (SEQ ID NO:16), NC_(—)003030) and E. coli (GenBank NOs:NP_(—)417484, NC_(—)000913).

The term “acetolactate synthase”, also known as “acetohydroxy acidsynthase”, refers to a polypeptide (or polypeptides) having an enzymeactivity that catalyzes the conversion of two molecules of pyruvic acidto one molecule of alpha-acetolactate. Acetolactate synthase, known asEC 2.2.1.6 [formerly 4.1.3.18] (Enzyme Nomenclature 1992, AcademicPress, San Diego) may be dependent on the cofactor thiamin pyrophosphatefor its activity. Suitable acetolactate synthase enzymes are availablefrom a number of sources, for example, Bacillus subtilis (GenBank Nos:AAA22222 NCBI (National Center for Biotechnology Information) amino acidsequence (SEQ ID NO:42), L04470 NCBI nucleotide sequence (SEQ IDNO:41)), Klebsiella terrigena (GenBank Nos: AAA25055, L04507), andKlebsiella pneumoniae (GenBank Nos: AAA25079 (SEQ ID NO:20), M73842 (SEQID NO:19).

The term “acetolactate decarboxylase” refers to a polypeptide (orpolypeptides) having an enzyme activity that catalyzes the conversion ofalpha-acetolactate to acetoin. Acetolactate decarboxylases are known asEC 4.1.1.5 and are available, for example, from Bacillus subtilis(GenBank Nos: AAA22223, L04470), Klebsiella terrigena (GenBank Nos:AAA25054, L04507) and Klebsiella pneumoniae (SEQ ID NO:18 (amino acid)SEQ ID NO:17 (nucleotide)).

The term “butanediol dehydrogenase” also known as “acetoin reductase”refers to a polypeptide (or polypeptides) having an enzyme activity thatcatalyzes the conversion of acetoin to 2,3-butanediol. Butanedioldehydrogenases are a subset of the broad family of alcoholdehydrogenases.

Butanediol dehydrogenase enzymes may have specificity for production ofR- or S-stereochemistry in the alcohol product. S-specific butanedioldehydrogenases are known as EC 1.1.1.76 and are available, for example,from Klebsiella pneumoniae (GenBank Nos: BBA13085 (SEQ ID NO:22),D86412. R-specific butanediol dehydrogenases are known as EC 1.1.1.4 andare available, for example, from Bacillus cereus (GenBank Nos.NP_(—)830481, NC_(—)004722; AAP07682, AE017000), and Lactococcus lactis(GenBank Nos. AAK04995, AE006323).

The term “butanediol dehydratase”, also known as “diol dehydratase” or“propanediol dehydratase” refers to a polypeptide (or polypeptides)having an enzyme activity that catalyzes the conversion of2,3-butanediol to 2-butanone, also known as methyl ethyl ketone (MEK).Butanediol dehydratase may utilize the cofactor adenosyl cobalamin.Adenosyl cobalamin-dependent enzymes are known as EC 4.2.1.28 and areavailable, for example, from Klebsiella oxytoca (GenBank Nos: BAA08099(alpha subunit) (SEQ ID NO:24), BAA08100 (beta subunit) (SEQ ID NO:26),and BBA08101 (gamma subunit) (SEQ ID NO:28), (Note all three subunitsare required for activity), D45071).

The term “2-butanol dehydrogenase” refers to a polypeptide (orpolypeptides) having an enzyme activity that catalyzes the conversion of2-butanone to 2-butanol. 2-butanol dehydrogenases are a subset of thebroad family of alcohol dehydrogenases. 2-butanol dehydrogenase may beNADH- or NADPH-dependent. The NADH-dependent enzymes are known as EC1.1.1.1 and are available, for example, from Rhodococcus ruber (GenBankNos: CAD36475 (SEQ ID NO:30), AJ491307 (SEQ ID NO:29)). TheNADPH-dependent enzymes are known as EC 1.1.1.2 and are available, forexample, from Pyrococcus furiosus (GenBank Nos: AAC25556, AF013169).

The term “acetohydroxy acid isomeroreductase” or “acetohydroxy acidreductoisomerase” refers to an enzyme that catalyzes the conversion ofacetolactate to 2,3-dihydroxyisovalerate using NADPH (reducednicotinamide adenine dinucleotide phosphate) as an electron donor.Preferred acetohydroxy acid isomeroreductases are known by the EC number1.1.1.86 and sequences are available from a vast array ofmicroorganisms, including, but not limited to, Escherichia coli (GenBankNos: NP_(—)418222 (SEQ ID NO:32), NC_(—)000913 (SEQ ID NO:31)),Saccharomyces cerevisiae (GenBank Nos: NP_(—)013459 (SEQ ID NO:44),NC_(—)001144 (SEQ ID NO:43)), Methanococcus maripaludis (GenBank Nos:CAF30210, BX957220), and Bacillus subtilis (GenBank Nos: CAB14789 (SEQID NO:46), Z99118 (SEQ ID NO:45)).

The term “acetohydroxy acid dehydratase” or “dihydroxy acid dehydratase”refers to an enzyme that catalyzes the conversion of2,3-dihydroxyisovalerate to α-ketoisovalerate. Preferred acetohydroxyacid dehydratases are known by the EC number 4.2.1.9. These enzymes areavailable from a vast array of microorganisms, including, but notlimited to, E. coli (GenBank Nos: YP_(—)026248 (SEQ ID NO:34),NC_(—)000913 (SEQ ID NO:33)), S. cerevisiae (GenBank Nos: NP_(—)012550(SEQ ID NO:48), NC_(—)001142 (SEQ ID NO:47)), M. maripaludis (GenBankNos: CAF29874, BX957219), and B. subtilis (GenBank Nos: CAB14105,Z99115).

The term “branched-chain α-keto acid decarboxylase” refers to an enzymethat catalyzes the conversion of α-ketoisovalerate to isobutyraldehydeand CO₂. Preferred branched-chain α-keto acid decarboxylases are knownby the EC number 4.1.1.72 and are available from a number of sources,including, but not limited to, Lactococcus lactis (GenBank Nos:AAS49166, AY548760; CAG34226 (SEQ ID NO:36), AJ746364, Salmonellatyphimurium (GenBank Nos: NP_(—)461346, NC_(—)003197), and Clostridiumacetobutylicum (GenBank Nos: NP_(—)149189, NC_(—)001988).

The term “branched-chain alcohol dehydrogenase” refers to an enzyme thatcatalyzes the conversion of isobutyraldehyde to isobutanol. Preferredbranched-chain alcohol dehydrogenases are known by the EC number1.1.1.265, but may also be classified under other alcohol dehydrogenases(specifically, EC 1.1.1.1 or 1.1.1.2). These enzymes utilize NADH(reduced nicotinamide adenine dinucleotide) and/or NADPH as electrondonor and are available from a number of sources, including, but notlimited to, S. cerevisiae (GenBank Nos: NP_(—)010656, NC_(—)001136;NP_(—)014051, NC_(—)001145), E. coli (GenBank Nos: NP_(—)417484 (SEQ IDNO:38), NC_(—)000913 (SEQ ID NO:37)), and C. acetobutylicum (GenBankNos: NP_(—)349892, NC_(—)003030).

The term “gene” refers to a nucleic acid fragment that is capable ofbeing expressed as a specific protein, optionally including regulatorysequences preceding (5′ non-coding sequences) and following (3′non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure.

As used herein the term “coding sequence” refers to a DNA sequence thatcodes for a specific amino acid sequence. “Suitable regulatorysequences” refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing site, effectorbinding site and stem-loop structure.

The term “promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of effecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

As used herein the term “transformation” refers to the transfer of anucleic acid fragment into a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The terms “plasmid” and “vector” refer to an extra chromosomal elementoften carrying genes which are not part of the central metabolism of thecell, and usually in the form of circular double-stranded DNA fragments.Such elements may be autonomously replicating sequences, genomeintegrating sequences, phage or nucleotide sequences, linear orcircular, of a single- or double-stranded DNA or RNA, derived from anysource, in which a number of nucleotide sequences have been joined orrecombined into a unique construction which is capable of introducing apromoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell. “Transformationvector” refers to a specific vector containing a foreign gene and havingelements in addition to the foreign gene that facilitates transformationof a particular host cell.

As used herein the term “codon degeneracy” refers to the nature in thegenetic code permitting variation of the nucleotide sequence withoutaffecting the amino acid sequence of an encoded polypeptide. The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA.

A “carbon substrate” means a carbon contain compound useful as an energysource of a yeast and may include but are not limited to monosaccharidessuch as glucose and fructose, oligosaccharides such as lactose orsucrose, polysaccharides such as starch or cellulose or mixtures thereofand unpurified mixtures from renewable feedstocks such as cheese wheypermeate, cornsteep liquor, sugar beet molasses, and barley malt.

A “cell having a reduced response in the general control response toamino acid starvation” refers herein to a cell that does not senseuncharged tRNA as a signal for induction of transcription of amino acidbiosynthetic genes, and/or it does not respond to amino acid starvationby inducing transcription of amino acid biosynthetic genes (Hinnebusch(2005) Ann. Rev. Microbiol. 59:407-450).

As used herein, an “isolated nucleic acid fragment” or “isolated nucleicacid molecule” will be used interchangeably and will mean a polymer ofRNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

A nucleic acid fragment is “hybridizable” to another nucleic acidfragment, such as a cDNA, genomic DNA, or RNA molecule, when asingle-stranded form of the nucleic acid fragment can anneal to theother nucleic acid fragment under the appropriate conditions oftemperature and solution ionic strength. Hybridization and washingconditions are well known and exemplified in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989),particularly Chapter 11 and Table 11.1 therein (entirely incorporatedherein by reference). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. Stringency conditionscan be adjusted to screen for moderately similar fragments (such ashomologous sequences from distantly related organisms), to highlysimilar fragments (such as genes that duplicate functional enzymes fromclosely related organisms). Post-hybridization washes determinestringency conditions. One set of preferred conditions uses a series ofwashes starting with 6×SSC, 0.5% SDS at room temperature for 15 min,then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and thenrepeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A morepreferred set of stringent conditions uses higher temperatures in whichthe washes are identical to those above except for the temperature ofthe final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C.Another preferred set of highly stringent conditions uses two finalwashes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringentconditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washeswith 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see Sambrooket al., supra, 9.50-9.51). For hybridizations with shorter nucleicacids, i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.7-11.8). In one embodimentthe length for a hybridizable nucleic acid is at least about 10nucleotides. Preferably a minimum length for a hybridizable nucleic acidis at least about 15 nucleotides; more preferably at least about 20nucleotides; and most preferably the length is at least aboutnucleotides. Furthermore, the skilled artisan will recognize that thetemperature and wash solution salt concentration may be adjusted asnecessary according to factors such as length of the probe.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: 1.) Computational MolecularBiology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.)Biocomputinq: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991).

Preferred methods to determine identity are designed to give the bestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the MegAlign™ program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequencesis performed using the “Clustal method of alignment” which encompassesseveral varieties of the algorithm including the “Clustal V method ofalignment” corresponding to the alignment method labeled Clustal V(described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in theMegAlign™ program of the LASERGENE bioinformatics computing suite(DNASTAR Inc.). For multiple alignments, the default values correspondto GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters forpairwise alignments and calculation of percent identity of proteinsequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2,GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of thesequences using the Clustal V program, it is possible to obtain a“percent identity” by viewing the “sequence distances” table in the sameprogram. Additionally the “Clustal W method of alignment” is availableand corresponds to the alignment method labeled Clustal W (described byHiggins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al.,Comput. Appl. Biosci. 8:189-191 (1992)) and found in the MegAlign™ v6.1program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.).Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTHPENALTY=0.2, Delay Divergen Seqs (%)=30, DNA Transition Weight=0.5,Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). Afteralignment of the sequences using the Clustal W program, it is possibleto obtain a “percent identity” by viewing the “sequence distances” tablein the same program.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides, from otherspecies, wherein such polypeptides have the same or similar function oractivity. Useful examples of percent identities include, but are notlimited to: 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentagefrom 70% to 100% may be useful in describing the present invention, suchas 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99%. Suitable nucleic acid fragments encode polypeptides with theabove identities and typically encode a polypeptide having at leastabout 250 amino acids, preferably at least 300 amino acids, and mostpreferably at least about 348 amino acids.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.,215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Altschul, S. F., et al.,J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten ormore contiguous amino acids or thirty or more nucleotides is necessaryin order to putatively identify a polypeptide or nucleic acid sequenceas homologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence. The instant specification teaches the complete amino acidand nucleotide sequence encoding particular alcohol dehydrogenaseproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

The invention encompasses more than the specific exemplary sequencesbecause it is well known in the art that alterations in an amino acidsequence or in a coding region wherein a chemically equivalent aminoacid is substituted at a given site, which does not effect thefunctional properties of the encoded protein, are common. For thepurposes of the present invention substitutions are defined as exchangeswithin one of the following five groups:

-   1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser,    Thr (Pro, Gly);-   2. Polar, negatively charged residues and their amides: Asp, Asn,    Glu, Gln;-   3. Polar, positively charged residues: His, Arg, Lys;-   4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys); and-   5. Large aromatic residues: Phe, Tyr, Trp.-   Thus, a codon for the amino acid alanine, a hydrophobic amino acid,    may be substituted by a codon encoding another less hydrophobic    residue (such as glycine) or a more hydrophobic residue (such as    valine, leucine, or isoleucine). Similarly, changes which result in    substitution of one negatively charged residue for another (such as    aspartic acid for glutamic acid) or one positively charged residue    for another (such as lysine for arginine) can also be expected to    produce a functionally equivalent product. In many cases, nucleotide    changes which result in alteration of the N-terminal and C-terminal    portions of the protein molecule would also not be expected to alter    the activity of the protein. Thus coding regions with the described    codon variations, and proteins with the described amino acid    variations are encompassed in the present invention.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.;Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989(hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. andEnquist, L. W. Experiments with Gene Fusions; Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y., 1984; and by Ausubel, F. M. etal., In Current Protocols in Molecular Biology, published by GreenePublishing and Wiley-Interscience, 1987. Additional methods used hereare in Methods in Enzymology, Volume 194, Guide to Yeast Genetics andMolecular and Cell Biology (Part A, 2004, Christine Guthrie and GeraldR. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.).

General Control Response Target Genes for Engineering Butanol Tolerancein Yeast

The invention relates to the discovery that reducing expression of agene involved in the general control response to amino acid starvationin Saccharomyces cerevisiae results in increased tolerance of cells tobutanol. The general control response to amino acid starvation in yeastis a complex system that senses the presence of uncharged tRNAs andresponds by inducing transcription of amino acid biosynthetic genes.This control system (reviewed in Hinebusch (2005) Ann. Rev. Microbiol.59: 407-450) includes genes that when mutated confer sensitivity to awide range of amino acid antagonists and analogs; these genes werecalled general control non-depressible, or GCN, for the mutant phenotypeof not responding to amino acid starvation.

For example, GCN2 encodes a protein (Gcn2p) which senses uncharged tRNAand binds to ribosomes via one Gcn2p domain, the carboxy-terminaldomain. Uncharged tRNA is sensed by a second internal domain of Gcn2ptermed His RS (for histidyl-tRNA synthetase like). This binding ofuncharged tRNA to the HRS domain results in yet another Gcn2p domain(PK) kinasing eukaryotic initiation factor 2 that is associated with GDP(eIF2˜GDP) producing eIF2-P˜GDP. In turn, eIF2-P˜GDP stimulatestranslation of the GCN4 encoded mRNA and Gcn4p (the GCN4 encodedprotein) activates expression of many genes involved in amino acidbiosynthesis.

Initiation of translation requires an activated form of an initiationfactor, eIF2: eIF2˜GTP. This activated form presents the initiatingtRNA, finet-tRNA, to the ribosome. eIF2-finet-tRNA˜GTP normally startstranslation by binding to ribosomes where eventually eIF2˜GDP isreleased. This form of the initiation factor is inactive and must beactivated by exchange of GTP for GDP producing eIF2-GTP. When Gcn2p'skinase is activated, eIF2˜GDP is hijacked yielding eIF2˜P. This form,eIF2˜P, blocks the Guanine Exchange Factor eIF2B from catalyzing thereaction: eIF2˜GDP+GTP−>eIF2˜GTP +GDP. Thus most translationalinitiation is retarded while translation of Gcn4p, the transcriptionalactivator of amino acid biosynthetic genes, is increased.

Additional GCN gene encoded proteins involved in the general controlresponse to amino acid starvation system in Saccharomyces cerevisiaeinclude:

-   Gcn1p: a positive regulator of the Gcn2p kinase activity-   Gcn3p: alpha subunit of the translation initiation factor eIF2B, a    positive regulator of GCN4 expression-   Gcn5p: histone acetyltransferase, acetylates N-terminal lysines on    histones H₂B and H3; catalytic subunit of the ADA and SAGA histone    acetyltransferase complexes-   Gcn6p: positive regulator of GCN4 transcription-   Gcn7p: positive regulator of GCN4 transcription-   Gcn8p: role undefined-   Gcn9p: role undefined-   Gcn20p: positive regulator of Gcn2p kinase activity, forms a complex    with Gcn1p

Given in Table 4 are the SEQ ID NOs for the Saccharomyces cerevisiaeGcn1-5p and Gcn20p proteins and their coding regions. Also given inTable 4 are representative coding regions and proteins for GCN genes ofYarrowia lipolytica and Candida albicans.

A mutation that reduces or eliminates expression of a protein involvedin the general control response to amino acid starvation in yeast willreduce the response and surprisingly provide an increase in butanoltolerance. Thus the present yeast host has a genetic modificationreducing activity of at least one protein involved in the generalcontrol response to amino acid starvation. Suitable genes for geneticmodification to reduce the general control response to amino acidstarvation include genes encoding Gcn1p, Gcn2p, Gcn3p, Gcn4p, Gcn5p,Gcn6p, Gcn7p, Gcn8p, Gcn9p, and Gcn20p. Examples of these proteins aregiven in Table 4 as SEQ ID NOS:50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, and 74. Genes encoding proteins with sequence identities of atleast about 80%, 85%, 90%, 95% or more to these proteins and having GCNactivity may be targets for genetic modification to reduce the generalcontrol response to amino acid starvation. More suitable targets aregenes encoding Gcn1p, Gcn2p, Gcn3p, Gcn4p, Gcn5p, and Gcn20p. Mostsuitable targets are genes encoding Gcn2p and Gcn4p.

Any yeast gene identified as encoding a Gcn1p, Gcn2p, Gcn3p, Gcn4p,Gcn5p, Gcn6p, Gcn7p, Gcn8p, Gcn9p, or Gcn20p protein, or other geneencoding a protein involved in the general control response to aminoacid starvation, is a target gene for modification in the correspondingyeast strain to create a strain of the present invention with increasedbutanol tolerance. Any type of yeast having a GCN system may beengineered for butanol tolerance using the method of the presentinvention. Yeast genera including Saccharomyces, Yarrowia, Candida, andHansenula have GCN systems (Bode et al. (199) J. Basic. Microbiol.30(1):31-5) and examples of GCN genes of Saccharomyces cerevisiae,Yarrowia lipolytica, and Candida albicans which are targets formodification to provide tolerance are listed in Table 4. Examples of GCNencoded proteins of Saccharomyces cerevisiae include SEQ ID NOs:50, 52,54, 56, 58, and 60. Examples of GCN encoded proteins of Yarrowialipolytica include SEQ ID NOs:62, 64, 66, and 68. Examples of GCNencoded proteins of Candida albicans include SEQ ID NOs:70, 72, and 74.In addition, homologs of GCN2 and GCN4 have been found in the moldNeurospora crassa (Paluh et al. (1988) Proc. Natl. Acad. Sci. USA85(11):3728-3732).

Other GCN system target genes may be identified in the literature and inbioinformatics databases well known to the skilled person. Additionally,the sequences described herein or those recited in the art may be usedto identify other homologs in nature. For example each of the GCNnucleic acid fragments described herein may be used to isolate genesencoding homologous proteins from the same or other yeasts. Isolation ofhomologous genes using sequence-dependent protocols is well known in theart. Examples of sequence-dependent protocols include, but are notlimited to: 1.) methods of nucleic acid hybridization; 2.) methods ofDNA and RNA amplification, as exemplified by various uses of nucleicacid amplification technologies [e.g., polymerase chain reaction (PCR),Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR),Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or stranddisplacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci.U.S.A., 89:392 (1992)]; and 3.) methods of library construction andscreening by complementation.

For example, genes encoding similar proteins or polypeptides to the GCNgenes described herein could be isolated directly by using all or aportion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired yeast using methodology wellknown to those skilled in the art. Specific oligonucleotide probes basedupon the disclosed nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis, supra). Moreover, theentire sequences can be used directly to synthesize DNA probes bymethods known to the skilled artisan (e.g., random primers DNA labeling,nick translation or end-labeling techniques), or RNA probes usingavailable in vitro transcription systems. In addition, specific primerscan be designed and used to amplify a part of (or full-length of) theinstant sequences. The resulting amplification products can be labeleddirectly during amplification reactions or labeled after amplificationreactions, and used as probes to isolate full-length DNA fragments underconditions of appropriate stringency. Heterologous genes may also beidentified using functional selections as illustrated by complementationselection for GCN function described in Paluh et al. (ibid.).

Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown in the art (Thein and Wallace, “The use of oligonucleotides asspecific hybridization probes in the Diagnosis of Genetic Disorders”, inHuman Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp33-50, IRL: Herndon, Va.; and Rychlik, W., In Methods in MolecularBiology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols:Current Methods and Applications. Humania: Totowa, N.J.).

Generally two short segments of the described sequences may be used inpolymerase chain reaction protocols to amplify longer nucleic acidfragments encoding homologous genes from DNA or RNA. The polymerasechain reaction may also be performed on a library of cloned nucleic acidfragments wherein the sequence of one primer is derived from thedescribed nucleic acid fragments, and the sequence of the other primertakes advantage of the presence of the polyadenylic acid tracts to the3′ end of the mRNA precursor encoding microbial genes.

Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) togenerate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (e.g., BRL,Gaithersburg, Md.), specific 3′ or 5′ cDNA fragments can be isolated(Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217(1989)).

Alternatively, the described GCN sequences may be employed ashybridization reagents for the identification of homologs. The basiccomponents of a nucleic acid hybridization test include a probe, asample suspected of containing the gene or gene fragment of interest,and a specific hybridization method. Probes are typicallysingle-stranded nucleic acid sequences that are complementary to thenucleic acid sequences to be detected. Probes are “hybridizable” to thenucleic acid sequence to be detected. The probe length can vary from 5bases to tens of thousands of bases, and will depend upon the specifictest to be done. Typically a probe length of about 15 bases to about 30bases is suitable. Only part of the probe molecule need be complementaryto the nucleic acid sequence to be detected. In addition, thecomplementarity between the probe and the target sequence need not beperfect. Hybridization does occur between imperfectly complementarymolecules with the result that a certain fraction of the bases in thehybridized region are not paired with the proper complementary base.

Hybridization methods are well defined. Typically the probe and samplemust be mixed under conditions that will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration,the shorter the hybridization incubation time needed. Optionally, achaotropic agent may be added. The chaotropic agent stabilizes nucleicacids by inhibiting nuclease activity. Furthermore, the chaotropic agentallows sensitive and stringent hybridization of short oligonucleotideprobes at room temperature (Van Ness and Chen, Nucl. Acids Res.19:5143-5151 (1991)). Suitable chaotropic agents include guanidiniumchloride, guanidinium thiocyanate, sodium thiocyanate, lithiumtetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate,potassium iodide and cesium trifluoroacetate, among others. Typically,the chaotropic agent will be present at a final concentration of about 3M. If desired, one can add formamide to the hybridization mixture,typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 Mbuffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), orbetween 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal),polyvinylpyrrolidone (about 250-500 kdal) and serum albumin. Alsoincluded in the typical hybridization solution will be unlabeled carriernucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g.,calf thymus or salmon sperm DNA, or yeast RNA), and optionally fromabout 0.5 to 2% wt/vol glycine. Other additives may also be included,such as volume exclusion agents that include a variety of polarwater-soluble or swellable agents (e.g., polyethylene glycol), anionicpolymers (e.g., polyacrylate or polymethylacrylate) and anionicsaccharidic polymers (e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formats.One of the most suitable is the sandwich assay format. The sandwichassay is particularly adaptable to hybridization under non-denaturingconditions. A primary component of a sandwich-type assay is a solidsupport. The solid support has adsorbed to it or covalently coupled toit immobilized nucleic acid probe that is unlabeled and complementary toone portion of the sequence.

Alternatively, because GCN sequences are well known, and becausesequencing of the genomes of fungi is prevalent (10 are completed, 71others have been subjected to a whole genome shotgun approach and arebeing assembled while 42 others are in progress), suitable GCN systemtarget genes may be identified on the basis of sequence similarity usingbioinformatics approaches alone, which are well known to one skilled inthe art.

Genetic Modification of General Control Response Genes in Yeast forButanol Tolerance

Many methods for genetic modification of target genes are known to oneskilled in the art and may be used to create the present yeast strains.Modifications that may be used to reduce or eliminate expression of atarget protein are disruptions that include, but are not limited to,deletion of the entire gene or a portion of the gene encoding a Gcnp,inserting a DNA fragment into a GCN gene (in either the promoter orcoding region) so that the protein is not expressed or expressed atlower levels, introducing a mutation into a GCn coding region which addsa stop codon or frame shift such that a functional protein is notexpressed, and introducing one or more mutations into a GCN codingregion to alter amino acids so that a non-functional or a lessenzymatically active protein is expressed. In addition, expression of aGCN gene may be blocked by expression of an antisense RNA or aninterfering RNA, and constructs may be introduced that result incosuppression. Moreover, a GCN gene may be synthesized whose expressionis low because rare codons are substituted for plentiful ones, and thisgene substituted for the endogenous corresponding GCN gene. Such a genewill produce the same polypeptide but at a lower rate. In addition, thesynthesis or stability of the transcript may be lessened by mutation.Similarly the efficiency by which a protein is translated from mRNA maybe modulated by mutation. All of these methods may be readily practicedby one skilled in the art making use of the known sequences encoding Gcnproteins. Yeast GCN sequences are publicly available, and representativesequences are listed in Table 4. One skilled in the art may choosespecific modification strategies to eliminate or lower the expression ofa GCN gene as desired to increase butanol tolerance.

DNA sequences surrounding a GCN coding sequence are also useful in somemodification procedures and are available for yeasts such as forSaccharomycse cerevisiae in the complete genome sequence coordinated byGenome Project ID9518 of Genome Projects coordinated by NCBI (NationalCenter for Biotechnology Information) with identifying GOPID #13838.Additional examples of yeast genomic sequences include that of Yarrowialipolytica, GOPIC #13837, and of Candida albicans, which is included inGPID #10771, #10701 and #16373. Other yeast genomic sequences can bereadily found by one of skill in the art in publicly availabledatabases.

In particular, DNA sequences surrounding a GCN coding sequence areuseful for modification methods using homologous recombination. Forexample, in this method GCN gene flanking sequences are placed boundinga selectable marker gene to mediate homologous recombination whereby themarker gene replaces the GCN gene. Also partial GCN gene sequences andGCN flanking sequences bounding a selectable marker gene may be used tomediate homologous recombination whereby the marker gene replaces aportion of the target GCN gene. In addition, the selectable marker maybe bounded by site-specific recombination sites, so that followingexpression of the corresponding site-specific recombinase, theresistance gene is excised from the GCN gene without reactivating thelatter. The site-specific recombination leaves behind a recombinationsite which disrupts expression of the Gcn protein. The homologousrecombination vector may be constructed to also leave a deletion in theGCN gene following excision of the selectable marker, as is well knownto one skilled in the art.

Deletions may be made using mitotic recombination as described in Wachet al. ((1994) Yeast 10:1793-1808). This method involves preparing a DNAfragment that contains a selectable marker between genomic regions thatmay be as short as 20 bp, and which bound a target DNA sequence. ThisDNA fragment can be prepared by PCR amplification of the selectablemarker gene using as primers oligonucleotides that hybridize to the endsof the marker gene and that include the genomic regions that canrecombine with the yeast genome. The linear DNA fragment can beefficiently transformed into yeast and recombined into the genomeresulting in gene replacement including with deletion of the target DNAsequence (as described in Methods in Enzymology, v194, pp 281-301(1991)).

Moreover, promoter replacement methods may be used to exchange theendogenous transcriptional control elements allowing another means tomodulate expression such as described in Mnaimneh et al. ((2004) Cell118(1):31-44).

Butanol Tolerance of the Present Modified Yeast Strain

A yeast strain of the present invention that is genetically modified forreduced response in the general control response for amino acidstarvation has improved tolerance to butanol. The tolerance of reducedresponse strains may be assessed by assaying their growth inconcentrations of butanol that are detrimental to growth of the parental(prior to genetic modification) strains. Improved tolerance is tobutanol compounds including 1-butanol, isobutanol, and 2-butanol. Theamount of tolerance observed will vary depending on the inhibitingchemical and its concentration, growth conditions, growth period, andthe specific genetically modified strain. For example, as shown inExample 1 herein, improved tolerance was observed with growth in 1%-2%isobutanol for 8 hours in a medium lacking amino acids other thanhistidine and leucine. In this medium the cells have more biosyntheticdemand than is the case in rich medium, which contains histidine andleucine. Other conditions for demonstration of the improved butanoltolerance of the present yeast strains include conditions wherebiosynthetic demand is higher than in rich medium conditions, includinga lack of any metabolic product, such as other amino acids, nucleotides,or fatty acids. Additionally the presence of inhibitors, osmoticimbalance, or other non-ideal growth conditions may provide conditionsfor demonstration of improved butanol tolerance.

Butanol Biosynthetic Pathway

In the present invention, a genetic modification conferring increasedbutanol tolerance, as described above, is engineered in a yeast cellthat is engineered to express a butanol biosynthetic pathway. Eithergenetic modification may take place prior to the other.

The butanol biosynthetic pathway may be a 1-butanol, 2-butanol, orisobutanol biosynthetic pathway. Particularly suitable yeast hosts forthe production of butanol and modification of the general controlresponse to amino acid starvation for increased butanol toleranceinclude, but are not limited to, members of the genera Saccharomyces,Candida, Hansenula and Yarowia. Preferred hosts include Saccharomycescerevesiae, Candida albicans and Yarowia lipolytica.

1-Butanol Biosynthetic Pathway

A biosynthetic pathway for the production of 1-butanol is described byDonaldson et al. in co-pending and commonly owned U.S. PatentApplication Publication No. 0080182308, incorporated herein byreference. This biosynthetic pathway comprises the following substrateto product conversions:

-   -   a) acetyl-CoA to acetoacetyl-CoA, as catalyzed for example by        acetyl-CoA acetyltransferase with protein sequence such as SEQ        ID NO:2, 4 or 40 encoded by the genes given as SEQ ID NO:1, 3 or        39;    -   b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, as catalyzed for        example by 3-hydroxybutyryl-CoA dehydrogenase with protein        sequence such as SEQ ID NO:6 encoded by the gene given as SEQ ID        NO:5;    -   c) 3-hydroxybutyryl-CoA to crotonyl-CoA, as catalyzed for        example by crotonase with protein sequence such as SEQ ID NO:8        encoded by the gene given as SEQ ID NO:7;    -   d) crotonyl-CoA to butyryl-CoA, as catalyzed for example by        butyryl-CoA dehydrogenase with protein sequence such as SEQ ID        NO:10 encoded by the gene given as SEQ ID NO:9;    -   e) butyryl-CoA to butyraldehyde, as catalyzed for example by        butyraldehyde dehydrogenase with protein sequence such as SEQ ID        NO:12 encoded by the gene given as SEQ ID NO:11; and    -   f) butyraldehyde to 1-butanol, as catalyzed for example by        1-butanol dehydrogenase with protein sequence such as SEQ ID        NO:14 or 16 encoded by the genes given as SEQ ID NO:13 or 15.

The pathway requires no ATP and generates NAD⁺ and/or NADP⁺, thus, itbalances with the central, metabolic routes that generate acetyl-CoA.

2-Butanol Biosynthetic Pathway

Biosynthetic pathways for the production of 2-butanol are described byDonaldson et al. in co-pending and commonly owned U.S. PatentApplication Publication Nos. 20070259410 and 20070292927, eachincorporated herein by reference. One 2-butanol biosynthetic pathwaycomprises the following substrate to product conversions:

-   -   a) pyruvate to alpha-acetolactate, as catalyzed for example by        acetolactate synthase with protein sequence such as SEQ ID NO:20        encoded by the gene given as SEQ ID NO:19;    -   b) alpha-acetolactate to acetoin, as catalyzed for example by        acetolactate decarboxylase with protein sequence such as SEQ ID        NO:18 encoded by the gene given as SEQ ID NO:17;    -   c) acetoin to 2,3-butanediol, as catalyzed for example by        butanediol dehydrogenase with protein sequence such as SEQ ID        NO:22 encoded by the gene given as SEQ ID NO:21;    -   d) 2,3-butanediol to 2-butanone, catalyzed for example by        butanediol dehydratase with protein sequence such as SEQ ID        NO:24, 26, or 28 encoded by genes given as SEQ ID NO:23, 25, or        27; and    -   e) 2-butanone to 2-butanol, as catalyzed for example by        2-butanol dehydrogenase with protein sequence such as SEQ ID        NO:30 encoded by the gene given as SEQ ID NO:29.        Isobutanol Biosynthetic Pathway

Biosynthetic pathways for the production of isobutanol are described byMaggio-Hall et al. in copending and commonly owned U.S. PatentApplication Publication No. 20070092957, incorporated herein byreference. One isobutanol biosynthetic pathway comprises the followingsubstrate to product conversions:

-   -   a) pyruvate to acetolactate, as catalyzed for example by        acetolactate synthase with protein sequence such as SEQ ID NO:20        or 42 encoded by genes given as SEQ ID NO:19 or 41;    -   b) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed for        example by acetohydroxy acid isomeroreductase with protein        sequence such as SEQ ID NO:32, 44 or 46 encoded by genes given        as SEQ ID NO:31, 43 or 45;    -   c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, as catalyzed        for example by acetohydroxy acid dehydratase with protein        sequence such as SEQ ID NO:34 encoded by the gene given as SEQ        ID NO:33; or dihydroxyacid dehydratase with protein sequence        such as SEQ ID NO:48 encoded by the gene given as SEQ ID NO:47;    -   d) α-ketoisovalerate to isobutyraldehyde, as catalyzed for        example by a branched-chain keto acid decarboxylase with protein        sequence such as SEQ ID NO:36 encoded by the gene given as SEQ        ID NO:35; and    -   e) isobutyraldehyde to isobutanol, as catalyzed for example by a        branched-chain alcohol dehydrogenase with protein sequence such        as SEQ ID NO:38 encoded by the gene given as SEQ ID NO:37.        Construction of Yeast Strains for Butanol Production

Any yeast strain that is genetically modified for butanol tolerance asdescribed herein is additionally genetically modified (before or aftermodification to tolerance) to incorporate a butanol biosynthetic pathwayby methods well known to one skilled in the art. Genes encoding theenzyme activities described above, or homologs that may be identifiedand obtained by commonly used methods, such as those described above,that are well known to one skilled in the art, are introduced into ayeast host. Representative coding and amino acid sequences for pathwayenzymes that may be used are given in Tables 1, 2, and 3, with SEQ IDNOs:1-48.

Methods for gene expression in yeasts are known in the art;specifically, basic yeast molecular biology protocols includingtransformation, cell growth, gene expression, gap repair recombination,etc. are described in Methods in Enzymology, Volume 194, Guide to YeastGenetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrieand Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.Expression of a gene in yeast typically requires a promoter, followed bythe coding region of interest, and a transcriptional terminator, all ofwhich are operably linked to provide expression cassettes. A number ofyeast promoters can be used in constructing expression cassettes forgenes encoding a butanol biosynthetic pathway, including, but notlimited to constitutive promoters FBA, GPD, and GPM, and the induciblepromoters GAL1, GAL10, and CUP1. Suitable transcriptional terminatorsinclude, but are not limited to FBAt, GPDt, GPMt, ERG10t, and GAL1t. Forexample, suitable promoters, transcriptional terminators, and the genesof a 1-butanol or isobutanol biosynthetic pathway may be cloned into E.coli-yeast shuttle vectors, as described in Example 2.

Typically used plasmids in yeast are shuttle vectors pRS423, pRS424,pRS425, and pRS426 (American Type Culture Collection, Rockville, Md.),which contain an E. coli replication origin (e.g., pMB1), a yeast 2μorigin of replication, and a marker for nutritional selection. Theselection markers for these four vectors are His3 (vector pRS423), Trp1(vector pRS424), Leu2 (vector pRS425) and Ura3 (vector pRS426). Thesevectors allow strain propagation in both E. coli and yeast strains.Typical hosts for gene cloning and expression include a yeast haploidstrain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) (Research Genetics,Huntsville, Ala., also available from ATCC 201388) and a diploid strainBY4743 (MATa/alpha his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0ura3Δ0/ura3Δ0) (Research Genetics, Huntsville, Ala., also available fromATCC 201390). Construction of expression vectors for genes encodingbutanol biosynthetic pathway enzymes may be performed by either standardmolecular cloning techniques in E. coli or by the gap repairrecombination method in yeast.

The gap repair cloning approach takes advantage of the highly efficienthomologous recombination in yeast. Typically, a yeast vector DNA isdigested (e.g., in its multiple cloning site) to create a “gap” in itssequence. A number of insert DNAs of interest are generated that containa ≧21 bp sequence at both the 5′ and the 3′ ends that sequentiallyoverlap with each other, and with the 5′ and 3′ terminus of the vectorDNA. For example, to construct a yeast expression vector for “Gene X’, ayeast promoter and a yeast terminator are selected for the expressioncassette. The promoter and terminator are amplified from the yeastgenomic DNA, and Gene X is either PCR amplified from its source organismor obtained from a cloning vector comprising Gene X sequence. There isat least a 21 bp overlapping sequence between the 5′ end of thelinearized vector and the promoter sequence, between the promoter andGene X, between Gene X and the terminator sequence, and between theterminator and the 3′ end of the linearized vector. The “gapped” vectorand the insert DNAs are then co-transformed into a yeast strain andplated on the medium containing the appropriate compound mixtures thatallow complementation of the nutritional selection markers on theplasmids. The presence of correct insert combinations can be confirmedby PCR mapping using plasmid DNA prepared from the selected cells. Theplasmid DNA isolated from yeast (usually low in concentration) can thenbe transformed into an E. coli strain, e.g. TOP10, followed by minipreps and restriction mapping to further verify the plasmid construct.Finally the construct can be verified by sequence analysis. Yeasttransformants of positive plasmids are grown for performing enzymeassays to characterize the activities of the enzymes expressed by thegenes of interest.

Fermentation Media

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include but are not limited tomonosaccharides such as glucose and fructose, oligosaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, or methanol for which metabolicconversion into key biochemical intermediates has been demonstrated. Inaddition to one and two carbon substrates methylotrophic organisms arealso known to utilize a number of other carbon containing compounds suchas methylamine, glucosamine and a variety of amino acids for metabolicactivity. For example, methylotrophic yeast are known to utilize thecarbon from methylamine to form trehalose or glycerol (Bellion et al.,Microb. Growth C1Compd., [Int. Symp.], 7th (1993), 415-32. Editor(s):Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK).Similarly, various species of Candida will metabolize alanine or oleicacid (Sutter et al., Arch. Microbiol. 153:485-489 (1990)). Hence it iscontemplated that the source of carbon utilized in the present inventionmay encompass a wide variety of carbon containing substrates and willonly be limited by the choice of organism.

Although it is contemplated that all of the above mentioned carbonsubstrates and mixtures thereof are suitable in the present invention,preferred carbon substrates are glucose, fructose, and sucrose.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of the enzymatic pathway necessary forbutanol production.

Culture Conditions

Typically cells are grown at a temperature in the range of about 20° C.to about 37° C. in an appropriate medium. Suitable growth media in thepresent invention are common commercially prepared media such as broththat includes yeast nitrogen base, ammonium sulfate, and dextrose as thecarbon/energy source) or YPD Medium, a blend of peptone, yeast extract,and dextrose in optimal proportions for growing most Saccharomycescerevisiae strains. Other defined or synthetic growth media may also beused and the appropriate medium for growth of the particularmicroorganism will be known by one skilled in the art of microbiology orfermentation science.

Suitable pH ranges for the fermentation are between pH 3.0 to pH 7.5,where pH 4.5.0 to pH 6.5 is preferred as the initial condition.

Fermentations may be performed under aerobic or anaerobic conditions,where anaerobic or microaerobic conditions are preferred.

The amount of butanol produced in the fermentation medium can bedetermined using a number of methods known in the art, for example, highperformance liquid chromatography (HPLC) or gas chromatography (GC).

Methods for Butanol Isolation from the Fermentation Medium

The bioproduced butanol may be isolated from the fermentation mediumusing methods known in the art. For example, solids may be removed fromthe fermentation medium by centrifugation, filtration, decantation, orthe like. Then, the butanol may be isolated from the fermentationmedium, which has been treated to remove solids as described above,using methods such as distillation, liquid-liquid extraction, ormembrane-based separation. Because butanol forms a low boiling point,azeotropic mixture with water, distillation can only be used to separatethe mixture up to its azeotropic composition. Distillation may be usedin combination with another separation method to obtain separationaround the azeotrope. Methods that may be used in combination withdistillation to isolate and purify butanol include, but are not limitedto, decantation, liquid-liquid extraction, adsorption, andmembrane-based techniques. Additionally, butanol may be isolated usingazeotropic distillation using an entrainer (see for example Doherty andMalone, Conceptual Design of Distillation Systems, McGraw Hill, NewYork, 2001).

The butanol-water mixture forms a heterogeneous azeotrope so thatdistillation may be used in combination with decantation to isolate andpurify the butanol. In this method, the butanol containing fermentationbroth is distilled to near the azeotropic composition. Then, theazeotropic mixture is condensed, and the butanol is separated from thefermentation medium by decantation. The decanted aqueous phase may bereturned to the first distillation column as reflux. The butanol-richdecanted organic phase may be further purified by distillation in asecond distillation column.

The butanol may also be isolated from the fermentation medium usingliquid-liquid extraction in combination with distillation. In thismethod, the butanol is extracted from the fermentation broth usingliquid-liquid extraction with a suitable solvent. The butanol-containingorganic phase is then distilled to separate the butanol from thesolvent.

Distillation in combination with adsorption may also be used to isolatebutanol from the fermentation medium. In this method, the fermentationbroth containing the butanol is distilled to near the azeotropiccomposition and then the remaining water is removed by use of anadsorbent, such as molecular sieves (Aden et al. Lignocellulosic Biomassto Ethanol Process Design and Economics Utilizing Co-Current Dilute AcidPrehydrolysis and Enzymatic Hydrolysis for Corn Stover, ReportNREL/TP-510-32438, National Renewable Energy Laboratory, June 2002).

Additionally, distillation in combination with pervaporation may be usedto isolate and purify the butanol from the fermentation medium. In thismethod, the fermentation broth containing the butanol is distilled tonear the azeotropic composition, and then the remaining water is removedby pervaporation through a hydrophilic membrane (Guo et al., J. Membr.Sci. 245, 199-210 (2004)).

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989)(Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, pub. by Greene Publishing Assoc. andWiley-Interscience (1987).

Materials and Methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following Examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989). All reagents, restriction enzymes andmaterials used for the growth and maintenance of bacterial cells wereobtained from Aldrich Chemicals (Milwaukee, Wis.), BD Diagnostic Systems(Sparks, Md.), Life Technologies (Rockville, Md.), or Sigma ChemicalCompany (St. Louis, Mo.) unless otherwise specified.

Microbial strains were obtained from The American Type CultureCollection (ATCC), Manassas, Va., unless otherwise noted.

Methods for Determining Isobutanol Concentration in Culture Media

The concentration of isobutanol in the culture media can be determinedby a number of methods known in the art. For example, a specific highperformance liquid chromatography (HPLC) method utilizes a ShodexSH-1011 column with a Shodex SH-G guard column, both purchased fromWaters Corporation (Milford, Mass.), with refractive index (RI)detection. Chromatographic separation is achieved using 0.01 M H₂SO₄ asthe mobile phase with a flow rate of 0.5 mL/min and a column temperatureof 50° C. Isobutanol has a retention time of 46.6 min under theconditions described. Alternatively, gas chromatography (GC) methods areavailable. For example, a specific GC method utilizes an HP-INNOWaxcolumn (30 m×0.53 mm id, 1 μm film thickness, Agilent Technologies,Wilmington, Del.), with a flame ionization detector (FID). The carriergas is helium at a flow rate of 4.5 mL/min, measured at 150° C. withconstant head pressure; injector split is 1:25 at 200° C.; oventemperature ias 45° C. for 1 min, 45 to 220° C. at 10° C./min, and 220°C. for 5 min; and FID detection is employed at 240° C. with 26 mL/minhelium makeup gas. The retention time of isobutanol is 4.5 min.

The meaning of abbreviations is as follows: “s” means second(s), “min”means minute(s), “h” means hour(s), “psi” means pounds per square inch,“nm” means nanometers, “d” means day(s), “μL” means microliter(s), “mL”means milliliter(s), “L” means liter(s), “mm” means millimeter(s), “nm”means nanometers, “mM” means millimolar, “μM” means micromolar, “M”means molar, “mmol” means millimole(s), “μmol” means micromole(s)”, “g”means gram(s), “μg” means microgram(s) and “ng” means nanogram(s), “PCR”means polymerase chain reaction, “OD” means optical density, “OD₆₀₀”means the optical density measured at a wavelength of 600 nm, “kDa”means kilodaltons, “g” means the gravitation constant, “bp” means basepair(s), “kbp” means kilobase pair(s), “% w/v” means weight/volumepercent, % v/v″ means volume/volume percent, “HPLC” means highperformance liquid chromatography, and “GC” means gas chromatography.The term “molar selectivity” is the number of moles of product producedper mole of sugar substrate consumed and is reported as a percent.

Example 1 Butanol tolerance in qcn2 and qcn4 mutants

GCN2 gene and GCN4 gene deletion mutants of the diploid a/αSaccharomycescerevisiae strain BY4743 (Brachmann et al. (Yeast 14:115-132 (1998)) areavailable in a nearly complete, ordered deletion strain collection(Giaever et al. Nature 418, 387-391 (2002); Saccharomyces GenomeDeletion Project). Cells of the GCN2 gene and GCN4 gene deletion mutantswere grown overnight from a single colony on a YPD plate in either YPDor YVCM medium (recipes below) in a 14 ml Falcon tube at 30° C. withshaking at 250 rpm. Overnight cultures were diluted 1:100 (2 ml to 200ml) in the same medium and growth was monitored every 60 minutes until 1doubling had occurred. At that point the cultures were split into 25 mlsamples that were dispensed to separate 125 ml plastic flasks.Challenging concentrations of isobutanol ranging between 0.5% and 2% w/vwere added to all but one flask of each culture that served as thepositive control. Control and challenge cultures were incubated withshaking in a 30° C. water bath and absorbance was monitored on about anhourly basis.

The two media used were a rich medium, YPD, which contains per liter: 10g yeast extract, 20 g peptone, 20 g dextrose; and a defined, syntheticmedium, YVCM, which contains per liter: 6.67 g yeast nitrogen basewithout amino acids but with ammonium sulfate, 20 g dextrose, 20 mgL-histidine, 30 mg L-leucine, 20 mg uracil.

Using 8 and 24 hr time points for growth in YVCM containing isobutanol,fractional growth yields were determined and results are given inFIG. 1. Both GCN2 and GCN4 deletion lines that were grown in thesynthetic medium were substantially more tolerant to an 8 hr isobutanolchallenge than the parental strain. The accrued advantage disappearedafter overnight incubation. The increased tolerance was seen over a 1-2%isobutanol concentration range.

Using 7 and 23 hr time points for growth in YPD containing isobutanol,fractional growth yields were determined and results are given in FIG.2. In these conditions improved tolerance was not observed at the shorttime point, and minimal improvement was seen with the GCN2 and GCN4mutations in different isobutanol concentrations.

Example 2 Expression of Isobutanol Pathway Genes in SaccharomycesCerevisiae

To express isobutanol pathway genes in Saccharomyces cerevisiae, anumber of E. coli-yeast shuttle vectors were constructed. A PCR approach(Yu, et al. Fungal Genet. Biol. 41:973-981 (2004)) was used to fusegenes with yeast promoters and terminators. Specifically, the GPDpromoter (SEQ ID NO:76) and CYC1 terminator (SEQ ID NO:77) were fused tothe aIsS gene from Bacillus subtilis (SEQ ID NO:41), the FBA promoter(SEQ ID NO:78) and CYC1 terminator were fused to the ILV5 gene from S.cerevisiae (SEQ ID NO:43), the ADH1 promoter (SEQ ID NO:79) and ADH1terminator (SEQ ID NO:80) were fused to the ILV3 gene from S. cerevisiae(SEQ ID NO:47), and the GPM promoter (SEQ ID NO:81) and ADH1 terminatorwere fused to the kivD gene from Lactococcus lactis (SEQ ID NO:35). Theprimers, given in Table 5, were designed to include restriction sitesfor cloning promoter/gene/terminator products into E. coli-yeast shuttlevectors from the pRS400 series (Christianson et al. Gene 110:119-122(1992)) and for exchanging promoters between constructs. Primers for the5′ ends of ILV5 and ILV3 (N138 and N155, respectively, given as SEQ IDNOs: 92 and 104, respectively) generated new start codons to eliminatemitochondrial targeting of these enzymes.

All fused PCR products were first cloned into pCR4-Blunt by TOPO cloningreaction (Invitrogen) and the sequences were confirmed (using M13forward and reverse primers (Invitrogen) and the sequencing primersprovided in Table 5. Two additional promoters (CUP1 and GAL1) werecloned by TOPO reaction into pCR4-Blunt and confirmed by sequencing;primer sequences are indicated in Table 5. The plasmids that wereconstructed are described in Table 6. The plasmids were transformed intoeither Saccharomyces cerevisiae BY4743 (ATCC 201390) or YJR148w (ATCC4036939) to assess enzyme specific activities. For the determination ofenzyme activities, cultures were grown to an OD₆₀₀ of 1.0 in syntheticcomplete medium (Methods in Yeast Genetics, 2005, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) lacking anymetabolite(s) necessary for selection of the expression plasmid(s),harvested by centrifugation (2600×g for 8 min at 4° C.), washed withbuffer, centrifuged again, and frozen at −80° C. The cells were thawed,resuspended in 20 mM Tris-HCl, pH 8.0 to a final volume of 2 mL, andthen disrupted using a bead beater with 1.2 g of glass beads (0.5 mmsize). Each sample was processed on high speed for 3 minutes total (withincubation on ice after each minute of beating). Extracts were clearedof cell debris by centrifugation (20,000×g for 10 min at 4° C.).

Acetolactate synthase activity in the cell free extracts is measuredusing the method described by Bauerle et al. (Biochim. Biophys. Acta92(1):142-149 (1964)). Acetohydroxy acid reductoisomerase activity inthe cell free extracts is measured using the method described by Arlinand Umbarger (J. Biol. Chem. 244(5):1118-1127 (1969)). Acetohydroxy aciddehydratase activity in the cell free extracts is measured using themethod described by Flint et al. (J. Biol. Chem. 268(20):14732-14742(1993)). Branched-chain keto acid decarboxylase activity in the cellfree extracts is measured using the method described by Smit et al.(Appl. Microbiol. Biotechnol. 64:396-402 (2003)), except that Purpald®reagent (Aldrich, Catalog No. 162892) is used to detect and quantify thealdehyde reaction products.

TABLE 5 Primer Sequences for Cloning and Sequencing ofS. cerevisiae Expression Vectors Name Sequence Description SEQ ID NO:N98SeqF1 CGTGTTAGTCACATCAGGAC B. subtilis alsS 82 sequencing primerN98SeqF2 GGCCATAGCAAAAATCCAAACAGC B. subtilis alsS 83 sequencing primerN98SeqF3 CCACGATCAATCATATCGAACACG B. subtilis alsS 84 sequencing primerN98SeqF4 GGTTTCTGTCTCTGGTGACG B. subtilis alsS 85 sequencing primerN99SeqR1 GTCTGGTGATTCTACGCGCAAG B. subtilis alsS 86 sequencing primerN99SeqR2 CATCGACTGCATTACGCAACTC B. subtilis alsS 87 sequencing primerN99SeqR3 CGATCGTCAGAACAACATCTGC B. subtilis alsS 88 sequencing primerN99SeqR4 CCTTCAGTGTTCGCTGTCAG B. subtilis alsS 89 sequencing primer N136CCGCGGATAGATCTGAAATGAA FBA promoter forward 90 TAACAATACTGACAprimer with SacII/BgIII sites N137 TACCACCGAAGTTGATTTGCTTCAFBA promoter reverse 91 ACATCCTCAGCTCTAGATTTGA primer with BbvCI siteATATGTATTACTTGGTTAT and ILV5-annealing region N138ATGTTGAAGCAAATCAACTTCGG ILV5 forward primer 92 TGGTA(creates alternate start codon) N139 TTATTGGTTTTCTGGTCTCAACILV5 reverse primer 93 N140 AAGTTGAGACCAGAAAACCAATA CYC terminator 94ATTAATTAATCATGTAATTAGTTA forward primer with TGTCACGCTTPacI site and ILV5- annealing region N141 GCGGCCGCCCGCAAATTAAAGCCCYC terminator 95 TTCGAGC reverse primer with NotI site N142GGATCCGCATGCTTGCATTTAGT GPM promoter forward 96 CGTGCprimer with BamHI site N143 CAGGTAATCCCCCACAGTATAC GPM promoter reverse97 ATCCTCAGCTATTGTAATATGTG primer with BbvCI site TGTTTGTTTGGand kivD-annealing region N144 ATGTATACTGTGGGGGATTACCkivD forward primer 98 N145 TTAGCTTTTATTTTGCTCCGCA kivD reverse primer99 N146 TTTGCGGAGCAAAATAAAAGCTAA ADH terminator 100TTAATTAAGAGTAAGCGAATTT forward primer with CTTATGATTTAPacI site and kivD- annealing region N147 ACTAGTACCACAGGTGTTGTCCTADH terminator 101 CTGAG reverse primer with SpeI site N151CTAGAGAGCTTTCGTTTTCATG alsS reverse primer 102 N152CTCATGAAAACGAAAGCTCTCTA CYC terminator 103 GTTAATTAATCATGTAATTAGTTAforward primer with TGTCACGCTT PacI site and alsS- annealing region N155ATGGCAAAGAAGCTCAACAAGT ILV3 forward primer 104 ACT(alternate start codon) N156 TCAAGCATCTAAAACACAACCG ILV3 reverse primer105 N157 AACGGTTGTGTTTTAGATGCTTG ADH terminator 106ATTAATTAAGAGTAAGCGAATTT forward primer with CTTAGATTTAPacI site and ILV3- annealing region N158 GGATCCTTTTCTGGCAACCAAACADH promoter forward 107 CCATA primer with BamHI site N159CGAGTACTTGTTGAGCTTCTTTG ADH promoter reverse 108 CCATCCTCAGCGAGATAGTTGAprimer with BbvCI site TTGTATGCTTG and ILV3-annealing region N160SeqF1GAAAACGTGGCATCCTCTC FBA::ILV5::CYC 109 sequencing primer N160SeqF2GCTGACTGGCCAAGAGAAA FBA::ILV5::CYC 110 sequencing primer N160SeqF3TGTACTTCTCCCACGGTTTC FBA::ILV5::CYC 111 sequencing primer N160SeqF4AGCTACCCAATCTCTATACCCA FBA::ILV5::CYC 112 sequencing primer N160SeqF5CCTGAAGTCTAGGTCCCTATTT FBA::ILV5::CYC 113 sequencing primer N160SeqR1GCGTGAATGTAAGCGTGAC FBA::ILV5::CYC 114 sequencing primer N160SeqR2CGTCGTATTGAGCCAAGAAC FBA::ILV5::CYC 115 sequencing primer N160SeqR3GCATCGGACAACAAGTTCAT FBA::ILV5::CYC 116 sequencing primer N160SeqR4TCGTTCTTGAAGTAGTCCAACA FBA::ILV5::CYC 117 sequencing primer N160SeqR5TGAGCCCGAAAGAGAGGAT FBA::ILV5::CYC 118 sequencing primer N161SeqF1ACGGTATACGGCCTTCCTT ADH::ILV3::ADH 119 sequencing primer N161SeqF2GGGTTTGAAAGCTATGCAGT ADH::ILV3::ADH 120 sequencing primer N161SeqF3GGTGGTATGTATACTGCCAACA ADH::ILV3::ADH 121 sequencing primer N161SeqF4GGTGGTACCCAATCTGTGATTA ADH::ILV3::ADH 122 sequencing primer N161SeqF5CGGTTTGGGTAAAGATGTTG ADH::ILV3::ADH 123 sequencing primer N161SeqF6AAACGAAAATTCTTATTCTTGA ADH::ILV3::ADH  124 sequencing primer N161SeqR1TCGTTTTAAAACCTAAGAGTCA ADH::ILV3::ADH 125 sequencing primer N161SeqR2CCAAACCGTAACCCATCAG ADH::ILV3::ADH 126 sequencing primer N161SeqR3CACAGATTGGGTACCACCA ADH::ILV3::ADH 127 sequencing primer N161SeqR4ACCACAAGAACCAGGACCTG ADH:ILV3::ADH 128 sequencing primer N161SeqR5CATAGCTTTCAAACCCGCT ADH::ILV3::ADH 129 sequencing primer N161SeqR6CGTATACCGTTGCTCATTAGAG ADH::ILV3::ADH 130 sequencing primer N162ATGTTGACAAAAGCAACAAAAGA alsS forward primer 131 N189ATCCGCGGATAGATCTAGTTCG GPD forward primer 132 AGTTTATCATTATCAAwith SacII/BglII sites N190.1 TTCTTTTGTTGCTTTTGTCAACATGPD promoter reverse 133 CCTCAGCGTTTATGTGTGTTTAT primer with BbvCI siteTCGAAA and alsS-annealing region N176 ATCCGCGGATAGATCTATTAGAAGAL1 promoter 134 GCGCCGAGCGGGCG forward primer with SacII/BglII sitesN177 ATCCTCAGCTTTTCTCCTTGACGT GAL1 promoter 135 TAAAGTAreverse with BbvCI site N191 ATCCGCGGATAGATCTCCCATTA CUP1 promoter 136CCGACATTTGGGCGC forward primer with SacII/BglII sites N192ATCCTCAGCGATGATTGATTGAT CUP1 promoter 137 TGATTGTAreverse with BbvCI site

TABLE 6 E. coli-Yeast Shuttle Vectors Carrying Isobutanol Pathway GenesPlasmid Name Construction pRS426 [ATCC No. 77107], — URA3 selectionpRS426::GPD::alsS::CYC GPD::alsS::CYC PCR product digested withSacII/NotI cloned into pRS426 digested with same pRS426::FBA::ILV5::CYCFBA::ILV5::CYC PCR product digested with SacII/NotI cloned into pRS426digested with same pRS425 [ATCC No. 77106], — LEU2 selectionpRS425::ADH::ILV3::ADH ADH::ILV3::ADH PCR product digested withBamHI/SpeI cloned into pRS425 digested with same pRS425::GPM::kivD::ADHGPM::kivD::ADH PCR product digested with BamHI/SpeI cloned into pRS425digested with same pRS426::CUP1::alsS 7.7 kbp SacII/BbvCI fragment frompRS426::GPD::alsS::CYC ligated with SacII/BbvCI CUP1 fragmentpRS426::GAL1::ILV5 7 kbp SacII/BbvCI fragment frompRS426::FBA::ILV5::CYC ligated with SacII/BbvCI GAL1 fragmentpRS425::FBA::ILV3 8.9 kbp BamHI/BbvCI fragment frompRS425::ADH::ILV3::ADH ligated with 0.65 kbp BglII/BbvCI FBA fragmentfrom pRS426::FBA::ILV5::CYC pRS425::CUP1-alsS + FBA-ILV3 2.4 kbpSacII/NotI fragment from pRS426::CUP1::alsS cloned intopRS425::FBA::ILV3 cut with SacII/NotI pRS426::FBA-ILV5 + GPM-kivD 2.7kbp BamHI/SpeI fragment from pRS425::GPM::kivD::ADH cloned intopRS426::FBA::ILV5::CYC cut with BamHI/SpeI pRS426::GAL1-FBA + GPM-kivD8.5 kbp SacII/NotI fragment from pRS426:: FBA- ILV5 + GPM-kivD ligatedwith 1.8 kbp SacII/NotI fragment from pRS426::GAL1::ILV5 pRS423 [ATCCNo. 77104], — HIS3 selection pRS423::CUP1-alsS + FBA-ILV3 5.2 kbpSacI/SalI fragment from pRS425::CUP1- alsS + FBA-ILV3 ligated intopRS423 cut with SacI/SalI pHR81 [ATCC No. 87541], — URA3 and leu2-dselection pHR81::FBA-ILV5 + GPM-kivD 4.7 kbp SacI/BamHI fragment frompRS426::FBA- ILV5 + GPM-kivD ligated into pHR81 cut with SacI/BamHI

Example 3 Prophetic Production of Isobutanol Using TolerantSaccharomyces cerevisiae Strain

The starting strain for this work is BY4741 (Brachmann, et al. Yeast.14: 115-132 (1998)) and its Δbat2 derivative, YJR148W BY4741, matingtype a (6939) available from the ATCC (#406939) with the genotype MATahis3delta1 leu2delta0 met15delta0 ura3delta0 deltaTWT2. bat2 encodes thecytosolic branched-chain amino acid aminotransferase, The deletion ofbat2 in combination with the URA3 deletion allows growth in the absenceof uracil to be used as a selection for the presence of a URA3insertion.

First ΔGCN2 and ΔGCN4 derivatives are made using the ATCC strain#406939. This is accomplished by a gene replacement strategy commonlyused in yeast in which a URA3⁺ allele is used as a selectable marker fora GCN insertion-deletion allele in which URA3⁺ is integrated in thegenome along with flanking direct repeat sequences replacing thesequence targeted for deletion. Subsequently a recombination eventbetween the direct repeats is selected by demanding fluoro-orotic acid(FOA) resistance which selects against URA3⁺ function.

The DNA fragment including a gene for URA3 expression and flankingdirect repeats (“URA3 repeats” fragment; SEQ ID NO:138) includes thefollowing (position numbers refer to position in the “URA3 repeats”fragment of SEQ ID NO:138):

-   1) primer binding sequences that bound the direct repeats flanking    URA3⁺: gcattgcggattacgtattctaatg (position 1-25; SEQ ID NO:143) and    gatgatacaacgagttagccaaggtg (position 1449-1474 of SEQ ID NO:144);-   2) the direct repeat sequences that flank the promoter and coding    sequence:    ttcagcccgcggaacgccagcaaatcaccacccatgcgcatgatactgagtcttgtacacgctgggcttcc    agtg (position 26-100 of SEQ ID NO:145) and    ttcagcccgcggaacgccagcaaatcaccacccatgcgcatgatactgagtcttgtacacgctgggcttcc    agtg (position 1375-1449 of SEQ ID NO:146)-   3) the promoter sequence:

ttttttattcttttttttgatttcggtttctttgaaatttttttgattcggtaatctccgaacagaaggaagaacgaaggaaggagcacagacttagattggtatatatacgcatatgtagtgttgaagaaacatgaaattgcccagtattcttaacccaactgcacagaacaaaaacctgcaggaaacgaagataaatc (position 149-348 of SEQ ID NO: 147) and

-   4) the coding region:

atgtcgaaagctacatataaggaacgtgctgctactcatcctagtcctgttgctgccaagctatttaatatcatgcacgaaaagcaaacaaacttgtgtgcttcattggatgttcgtaccaccaaggaattactggagttagttgaagcattaggtcccaaaatttgtttactaaaaacacatgtggatatcttgactgatttttccatggagggcacagttaagccgctaaaggcattatccgccaagtacaattttttactcttcgaagacagaaaatttgctgacattggtaatacagtcaaattgcagtactctgcgggtgtatacagaatagcagaatgggcagacattacgaatgcacacggtgtggtgggcccaggtattgttagcggtttgaagcaggcggcagaagaagtaacaaaggaacctagaggccttttgatgttagcagaattgtcatgcaagggctccctatctactggagaatatactaagggtactgttgacattgcgaagagcgacaaagattttgttatcggctttattgctcaaagagacatgggtggaagagatgaaggttacgattggttgattatgacacccggtgtgggtttagatgacaagggagacgcattgggtcaacagtatagaaccgtggatgatgtggtctctacaggatctgacattattattgttggaagaggactatttgcaaagggaagggatgctaaggtagagggtgaacgttacagaaaagcaggctgggaagcatatttgagaagatgcggccagcaaaactaa (position 349-1152 of SEQ ID NO: 148).

A DNA fragment containing a 50 bp sequence that is 100 bp upstream ofthe GCN2 coding region, the URA3 repeats fragment described above, and a50 bp sequence that is 100 bp downstream of the GCN2 coding region isprepared using PCR. The 5′ primer is a chimeric sequence containing 50bp of sequence upstream of GCN2 and the position 1-25 primer bindingsequence above in (1): 50 (GCN2 5′ flanking)+5′ ura3 primer (I) (SEQ IDNO:139). The 3′ primer is a chimeric sequence containing the complementof 50 bp of sequence downstream of GCN2 and the position 1449-1474primer binding sequence complement: 50 (reverse compl of GCN2 3′flanking)+3′ ura3 primer (reverse compl) (II) (SEQ ID NO:140).

The PCR reaction is a 50 μl reaction mixture of 1 μl of template DNA (50ng total), 1 μl of each primer at 20 μM, 25 μl of 2× TaKaRa Ex Taqpremix, 22 μl water. The template is pUC19-URA3 repeat, a pUC19(Yanisch-Perron et al. (1985) Gene, 33:103-119) derivative into whichthe “URA3 repeat” has been inserted at the multi-cloning site. The PCRcondition used is:

94° C. 1 min, then 30 cycles of 94° C. 20 sec, 55° C. 20 sec and 72° C.2 min, followed by 7 min at 72° C. The extension time is 1 min per kb.

The resulting PCR product, a ΔGCN2::URA3⁺ fragment, is purified using aQiagen PCR purification kit.

A similar DNA fragment is prepared as above but using primers containingsequences upstream and downstream of the GCN4 coding region: 50 (GCN4 5′flanking)+5′ ura3 primer (III) (SEQ ID NO:141) and 50 (reverse compl. ofGCN4 3′ flanking)+3′ ura3 primer (reverse compl) (GCN4) (IV) (SEQ IDNO:142).

The resulting PCR product, a ΔGCN4::URA3⁺ fragment, is purified using aQiagen PCR purification kit.

The PCR products are used to transform the strain ATCC #406939.Integrants are selected for growth in the absence of uracil. Integrantstrains with insertion of “URA3 repeats” and deletion of GCN2 or GCN4are called, respectively:

-   DYW1: MATa his3delta1 leu2delta0 met15delta0 ura3delta0 deltaTWT2    Δgcn2::URA3⁺ and-   DYW2: MATa his3delta1 leu2delta0 met15delta0 ura3delta0 deltaTWT2    Δgcn4::URA3⁺.

Using 5-FOA selection to select for elimination of the URA3⁺ allele,strains with recombination between the direct repeats are obtained andcalled:

-   DYW3: MATa his3delta1 leu2delta0 met15delta0 ura3delta0 deltaTWT2    Δgcn2 and-   DYW4: MATa his3delta1 leu2delta0 met15delta0 ura3delta0 deltaTWT2    Δgcn4

Plasmids pRS423::CUP1-alsS+FBA-ILV3 and pHR81::FBA -ILV5+ GPM-kivD(described in Example 2) are transformed into Saccharomyces cerevisiaeDYW3 and DYW4 to produce strains DYW3(Δgcn2)/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+ GPM-kivD and DYW4(Δgcn4)/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+ GPM-kivD. A controlstrain is prepared by transforming vectors pRS423 and pHR81 (describedin Example 2) into Saccharomyces cerevisiae (ATCC strain #406939)[strain 406939 (GCN2⁺ GCN4⁺)/pRS423/pHR811. Strains are maintained onstandard S. cerevisiae synthetic complete medium (Methods in YeastGenetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., pp. 201-202) containing either 2% glucose or sucrose but lackinguracil and histidine to ensure maintenance of plasmids.

For isobutanol production, cells are transferred to synthetic completemedium lacking uracil, histidine and leucine. Removal of leucine fromthe medium is intended to trigger an increase in copy number of thepHR81-based plasmid due to poor transcription of the leu2-d allele(Erhart and Hollenberg, J. Bacteriol. 156:625-635 (1983)). Aerobiccultures are grown in 175 mL capacity flasks containing 50 mL of mediumin an Innova4000 incubator (New Brunswick Scientific, Edison, N.J.) at30° C. and 200 rpm. Low oxygen cultures are prepared by adding 45 mL ofmedium to 60 mL serum vials that are sealed with crimped caps afterinoculation and kept at 30° C. Sterile syringes are used for samplingand addition of inducer, as needed. Approximately 24 h afterinoculation, the inducer CuSO₄ is added to a final concentration of 0.03mM. Control cultures for each strain without CuSO₄ addition are alsoprepared. Culture supernatants are analyzed 18 or 19 h and 35 h afterCuSO₄ addition by both HPLC (Shodex Sugar SH1011 column (Showa DenkoAmerica, Inc. NY) with refractive index (R1) detection) and GC (VarianCP-WAX 58(FFAP) CB, 0.25 mm×0.2 μm×25 m (Varian, Inc., Palo Alto,Calif.) with flame ionization detection (FID)) for isobutanol content,as described in the General Methods section. Production of isobutanol isenhanced by the presence of the mutant gcn alleles. In general, higherlevels of isobutanol per optical density unit are produced by the GCNmutants.

What is claimed is:
 1. A recombinant yeast host cell having thefollowing characteristics: a) the yeast host cell produces a butanolwhen grown in a medium containing a carbon substrate; b) the yeast hostcell comprises at least one genetic modification which reduces theresponse in the general control response to amino acid starvation,wherein the target for said genetic modification is a gene encoding theGeneral Control Nonderepressible (Gcn) protein Gcn2p; and c) the yeasthost cell comprises a recombinant biosynthetic pathway selected from thegroup consisting of: i) a 1-butanol biosynthetic pathway; ii) a2-butanol biosynthetic pathway; and iii) an isobutanol biosyntheticpathway.
 2. The yeast cell of claim 1, wherein the at least one geneticmodification reduces production of Gcn2p.
 3. The yeast cell of claim 2,wherein the at least one genetic modification is a disruption in anendogenous gene encoding.
 4. The yeast cell of claim 1, wherein the cellis a member of the genus Saccharomyces.
 5. The yeast of claim 1, wherethe cell is Saccharomyces cerevisiae comprising a disruption in anendogenous gene encoding.
 6. The recombinant yeast cell of claim 1,wherein the host cell comprises an isobutanol biosynthetic pathwaycomprising: a) at least one gene encoding an acetolactate synthase; b)at least one gene encoding acetohydroxy acid isomeroreductase; c) atleast one gene encoding acetohydroxy acid dehydratase; d) at least onegene encoding branched-chain keto acid decarboxylase; and e) at leastone gene encoding branched-chain alcohol dehydrogenase.
 7. A process forproduction of a butanol from recombinant yeast cell comprising: (a)providing the recombinant teast host cell of claim 1; (b) culturing thehost cell of step (a), wherein the butanol is produced; and (c)recovering the butanol made in step (b).
 8. The process of claim 7,wherein the host cell comprises an isobutanol biosynthetic pathwaycomprising: a) at least one gene encoding an acetolactate synthase; b)at least one gene encoding acetohydroxy acid isomeroreductase; c) atleast one gene encoding acetohydroxy acid dehydratase; d) at least onegene encoding branched-chain keto acid decarboxylase; and e) at leastone gene encoding branched-chain alcohol dehydrogenase.
 9. The processof claim 7, wherein the at least one genetic modification reducesproduction of Gcn2p.
 10. The recombinant yeast cell of claim 1, whereinthe yeast cell comprises a 1-butanol biosynthetic pathway comprising: a)at least one gene encoding an acetyl-CoA acetyltransferase; b) at leastone gene encoding a 3-hydroxybutyryl-CoA dehydrogenase; c) at least onegene encoding a crotonase; d) at least one gene encoding a butyryl-CoAdehydrogenase; e) at least one gene encoding a butyraldehydedehydrogenase; and f) at least one gene encoding a 1-butanoldehydrogenase.
 11. The recombinant yeast cell of claim 1, wherein theyeast cell comprises a 2-butanol biosynthetic pathway comprising: a) atleast one gene encoding an acetolactate synthase; b) at least one geneencoding an acetolactate decarboxylase; c) at least one gene encoding abutanediol dehydrogenase; d) at least one gene encoding a butanedioldehydratase; and e) at least one gene encoding a 2-butanoldehydrogenase.
 12. The process of claim 7, wherein the yeast host cellcomprises a 1-butanol biosynthetic pathway comprising: a) at least onegene encoding an acetyl-CoA acetyltransferase; b) at least one geneencoding a 3-hydroxybutyryl-CoA dehydrogenase; c) at least one geneencoding a crotonase; d) at least one gene encoding a butyryl-CoAdehydrogenase; e) at least one gene encoding a butyraldehydedehydrogenase; and f) at least one gene encoding a 1-butanoldehydrogenase.
 13. The process of claim 7, wherein the yeast host cellcomprises a 2-butanol biosynthetic pathway comprising: a) at least onegene encoding an acetolactate synthase; b) at least one gene encoding anacetolactate decarboxylase; c) at least one gene encoding a butanedioldehydrogenase; d) at least one gene encoding a butanediol dehydratase;and e) at least one gene encoding a 2-butanol dehydrogenase.